Encoded assays

Abstract

A method of conducting an assay for a set of targets, comprising: subjecting each target of a set of targets to a recognition event, in which each target is uniquely recognized by and bound to a recognition element associated with a code from a set of codes, thereby yielding a set of coded targets comprising the target and the recognition element; subjecting each recognition element of the set of coded targets to a transformation event, in which a molecular transformation of each recognition element produces a modified recognition element, thereby yielding a set of modified recognition elements comprising the code; subjecting each code of the set of modified recognition elements to an amplifying event, in which each code is amplified, thereby yielding a set of amplified codes; subjecting each amplified code of the set of amplified codes to a detection event, thereby decoding the code.

We claim: 
     
         1 . A method of conducting an assay for a set of targets, the method comprising:
 (a) subjecting a set of targets to a recognition event, in which each target is uniquely recognized by and bound to at least one recognition element from a set of coded recognition elements, each recognition element comprising a target-specific binding site and a code from a set of codes, wherein each code comprises at least one segment encoding one or more symbols, to yield a set of coded targets, wherein each coded target of the set of coded targets comprises a target of the set of targets bound to a recognition element of the set of coded recognition elements;   (b) subjecting the set of coded recognition elements of the set of coded targets to a molecular transformation event to yield a set of circularized coded recognition elements;   (c) performing rolling circle amplification of the circularized coded recognition elements to produce amplified circularized coded recognition elements; and   (d) detecting the set of targets associated with the amplified circularized coded recognition elements by decoding the amplified codes of the amplified circularized coded recognition elements, wherein the decoding comprises performing soft decision decoding.   
     
     
         2 . The method of  claim 1 , wherein the set of coded recognition elements comprises at least coded recognition elements. 
     
     
         3 . The method of  claim 1 , wherein the decoding the amplified codes comprises interrogating the at least one segment of the code with a method comprising nucleic acid sequencing or nucleic acid hybridization. 
     
     
         4 . The method of  claim 3 , wherein the nucleic acid sequencing comprises nanopore sequencing, Sanger sequencing, sequencing by synthesis, pyrosequencing, single molecule real-time sequencing, or sequencing by ligation. 
     
     
         5 . The method of  claim 1 , wherein the at least one segment of the code is interrogated by nucleic acid hybridization with one or more hybridization probes having at least one label to produce a detectable signal. 
     
     
         6 . The method of  claim 1 , wherein the code comprises a plurality of segments including the at least one segment, and wherein the code is interrogated by nucleic acid hybridization with a plurality of hybridization probes, wherein each hybridization probe comprises a label that is distinct from the other hybridization probes of the plurality to produce multiple detectable signals. 
     
     
         7 . The method of  claim 1 , wherein the molecular transformation event comprises a ligation reaction in which each coded recognition element of the set of coded targets is ligated to form the set of circularized coded recognition elements. 
     
     
         8 . The method of  claim 7 , wherein the subjecting the set of coded recognition elements of the set of coded targets to the molecular transformation event comprises introducing the set of coded recognition elements to a ligase enzyme under conditions sufficient to ligate and circularize the coded recognition elements. 
     
     
         9 . The method of  claim 1 , wherein the set of coded recognition elements comprises padlock probes or molecular inversion probes. 
     
     
         10 . The method of  claim 9 , wherein the set of coded recognition elements further comprises:
 (a) a 5′ probe arm and a 3′ probe arm; and   (b) a splint oligonucleotide probe comprising:
 (i) a 5′ region configured to bind to the 3′ probe arm; and 
 (ii) a 3′ region configured to bind to the 5′ probe arm. 
   
     
     
         11 . The method of  claim 1 , wherein the set of coded recognition elements further comprises:
 (a) one or more sequencing primer binding sequences;   (b) one or more amplification primer binding sequences;   (c) a unique identifier sequence (UMI);   (d) a sample index;   (e) a restriction enzyme site; or   (f) any combination of (a) to (e).   
     
     
         12 . The method of  claim 11 , wherein the one or more amplification primer binding sequences comprises a universal primer binding sequence that is common to all coded recognition elements of the set of coded recognition elements. 
     
     
         13 . The method of  claim 1 , wherein the rolling circle amplification event is performed on a surface. 
     
     
         14 . The method of  claim 1 , wherein the rolling circle amplification event is performed on a surface, wherein the surface comprises a cation-coating layer. 
     
     
         15 . The method of  claim 1 , wherein the rolling circle amplification event is performed on a surface, wherein the surface comprises a density of the amplified circularized coded recognition elements of about 23 thousand to about 110 thousand amplified circularized coded recognition elements per square millimeter (mm 2 ). 
     
     
         16 . The method of  claim 1 , further comprising condensing the amplified circularized coded recognition elements by addition of one or more condensing agents, wherein the one or more condensing agents comprises:
 (a) one or more cationic additives; or   (b) one or more multivalent oligonucleotide sequences configured to crosslink sites on the amplified circularized coded recognition elements.   
     
     
         17 . The method of  claim 14 , wherein the amplified circularized coded recognition elements comprise one or more modified nucleotides that participate in a crosslinking reaction with the one or more multivalent oligonucleotide sequences. 
     
     
         18 . The method of  claim 1 , wherein the set of codes are error corrected. 
     
     
         19 . The method of  claim 1 , wherein the set of codes are generated using 4-state encoding with 3 transitions per state. 
     
     
         20 . The method of  claim 1 , wherein each code of the set of codes has a length comprising 5 to 100 contiguous nucleotides. 
     
     
         21 . The method of  claim 1 , wherein each code comprises 3 or more segments including the at least one segment. 
     
     
         22 . The method of  claim 1 , wherein each code comprises at least 16 symbols including the one or more symbols. 
     
     
         23 . The method of  claim 1 , wherein the set of targets comprises hundreds or more targets. 
     
     
         24 . The method of  claim 1 , wherein the set of targets is obtained from a biological sample. 
     
     
         25 . The method of  claim 1 , wherein the set of targets comprises:
 (a) one or more substitutions, insertions and/or deletions; or   (b) a copy number variation.   
     
     
         26 . The method of  claim 1 , wherein the set of targets comprises one or more methylated nucleotides. 
     
     
         27 . The method of  claim 1 , wherein the set of targets comprises a pathogenic nucleic acid. 
     
     
         28 . The method of  claim 1 , wherein the subjecting the set of targets to the recognition event comprises introducing the set of targets at an input concentration of about 10 picomolar (pM) to about 100 pM to the set of coded recognition elements. 
     
     
         29 . The method of  claim 27 , wherein each coded recognition element of the set of coded recognition elements has a concentration of about 250 pM. 
     
     
         30 . The method of  claim 1 , further comprising introducing an exonuclease to the set of circularized coded recognition elements following the molecular transformation event prior to performing rolling circle amplification.